Table 1.

Automated extraction protocol on the system for sputum-to-genotype testing.

Operation	Programmed Parameter(s)
Sample homogenization	Rotate MagVor at 4950 rpm. 10 min *
Add sample to lysis buffer	Add 250 μL of sample (in two steps; 125 μL per transfer) to the 380 μL of lysis buffer in the heater strip. ~2.5 min
Heat incubation	10 min at 56°C
Add 95% ethanol	500 μL. ~1 min
Binding Step	20 pipetting cyclest†; 20 min
TruTip Wash 1	10 pipetting cycles; 7 min
TruTip Wash 2	5 pipetting cycles; ~2.5 min
TruTip Wash 3	5 pipetting cycles; ~2.5 min
Air dry TruTip matrix	90 sec forced air per TruTip; 10 min
Elute purified DNA	100 μL, 10 pipetting cycles; ~2 min
Add DNA to Master mix	Transfer 40 μL of DNA solution from one column on deep-well reagent plate to the next column with 92 μL of PCR mix‡; ~1 min
DNA-to-Genotype protocol starts here.
Add DNA to Master mix	Transfer 40 μL of DNA solution from one column on the deep-well reagent plate to the next column with 92 μL of PCR mix. ~1 mm
Mix	100 μL pipetting volume; 20 pipetting cycles; ~1.5 min
Transfer mix to LFA	Transfer 60 μL from deep-well reagent plate and introduce 52 μL of liquid through inlet ports of housing in order to fill LFA; ~1 mm
PCR	Lower thermal cycler onto LFA. PCR Protocol: initial denaturation for 5 min at 89°C; 30 cycles of 87°C for 50 s, 60 to 55°C (touchdown) for 65 s, and 65°C for 35 s; 20 cycles of 87°C for 50 s, 55°C for 65 s, and 65°C for 35 s; final extension at 65 °C for 3 min. Total time 3h 10 min.
Hybridization	54oC;3h
Wash	125 μL 3 times; ~4.5 min
Image and Analyze	Acquire image and analyze for each array. Move camera to each chamber, acquire image, analyze; ~5 min

^*TruTip was left off of lane with DNA sample (external positive control).

^†All pipetting operations were programmed at 135 μL sec⁻¹ flow rate.

^‡Tip was added to nozzel of lane with DNA sample (external positive control) to serve as a means of evaluating the DNA-to-genotype testing.